The significance of frontal plane static alignment in anticipating dynamic knee moment among transtibial prosthesis users: A cross-sectional study.

BACKGROUND: In the process of transtibial prosthetic fitting, alignment is the process of positioning the prosthetic foot relative to the residual limb. Changes in frontal plane alignment can impact knee moments during walking, which can either cause or, when aligned properly, prevent injuries. However, clinical evaluation of dynamic knee moments is challenging, limiting prosthetists' insights into dynamic joint loading. Typically, knee joint loading is assessed in static stance using the knee moment arm as a proxy for subsequent dynamic alignment. It remains uncertain if static alignment accurately represents actual dynamics during walking. RESEARCH QUESTION: Is the frontal knee moment arm in stance predictive for the knee moment arm and external knee adduction moment during gait in transtibial bone-anchored prosthesis users? METHODS: In this cross-sectional study, twenty-seven unilateral transtibial bone-anchored prosthesis users underwent data acquisition on the M-Gait instrumented treadmill. Static and dynamic measurements were conducted, and knee moment arm and external knee adduction moment were calculated. Pearson's correlation and linear regression analyses were performed to examine relationships between static and dynamic knee moment arms and external knee adduction moments. RESULTS: The static knee moment arm showed significant associations with dynamic knee moment arm at the ground reaction force peaks (First: r=0.60, r2=35%, p<0.001; Second: r=0.62, r2=38%, p=0.001) and knee adduction moment (First: r=0.42, r2=17%, p=0.030; Second: r=0.59, r2=35%, p=0.001). A 1 mm between-subject difference in static knee moment arm corresponded, on average, with a 0.9% difference in knee adduction moment at the first peak and a 1.5% difference at the second peak of the ground reaction force. SIGNIFICANCE: While static alignment is important to optimize adduction moments during stance it may only partly mitigate excessive moments during gait. The fair correlation and limited percentage of explained variance underscores the importance of dynamic alignment in optimizing the body's dynamic load during walking.

Collagen Tubular Airway-on-Chip for Extended Epithelial Culture and Investigation of Ventilation Dynamics.

The lower respiratory tract is a hierarchical network of compliant tubular structures that are made from extracellular matrix proteins with a wall lined by an epithelium. While microfluidic airway-on-a-chip models incorporate the effects of shear and stretch on the epithelium, week-long air-liquid-interface culture at physiological shear stresses, the circular cross-section, and compliance of native airway walls have yet to be recapitulated. To overcome these limitations, a collagen tube-based airway model is presented. The lumen is lined with a confluent epithelium during two-week continuous perfusion with warm, humid air while presenting culture medium from the outside and compensating for evaporation. The model recapitulates human small airways in extracellular matrix composition and mechanical microenvironment, allowing for the first time dynamic studies of elastocapillary phenomena associated with regular breathing and mechanical ventilation, as well as their impacts on the epithelium. A case study reveales increasing damage to the epithelium during repetitive collapse and reopening cycles as opposed to overdistension, suggesting expiratory flow resistance to reduce atelectasis. The model is expected to promote systematic comparisons between different clinically used ventilation strategies and, more broadly, to enhance human organ-on-a-chip platforms for a variety of tubular tissues.

Pseudomonas putida as a platform for medium-chain length α,ω-diol production: Opportunities and challenges.

Medium-chain-length α,ω-diols (mcl-diols) play an important role in polymer production, traditionally depending on energy-intensive chemical processes. Microbial cell factories offer an alternative, but conventional strains like Escherichia coli and Saccharomyces cerevisiae face challenges in mcl-diol production due to the toxicity of intermediates such as alcohols and acids. Metabolic engineering and synthetic biology enable the engineering of non-model strains for such purposes with P. putida emerging as a promising microbial platform. This study reviews the advancement in diol production using P. putida and proposes a four-module approach for the sustainable production of diols. Despite progress, challenges persist, and this study discusses current obstacles and future opportunities for leveraging P. putida as a microbial cell factory for mcl-diol production. Furthermore, this study highlights the potential of using P. putida as an efficient chassis for diol synthesis.

Lumbopelvic movement coordination during walking improves with transfemoral bone anchored limbs: Implications for low back pain.

BACKGROUND: Low back pain (LBP) is more prevalent in patients with transfemoral amputation using socket prostheses than able-bodied individuals, in part due to altered spinal loading caused by aberrant lumbopelvic movement patterns. Early evidence surrounding bone-anchored limb functional outcomes is promising, yet it remains unknown if this novel prosthesis influences LBP or movement patterns known to increase its risk. RESEARCH QUESTION: How are self-reported measures of LBP and lumbopelvic movement coordination patterns altered when using a unilateral transfemoral bone-anchored limb compared to a socket prosthesis? METHODS: Fourteen patients with unilateral transfemoral amputation scheduled to undergo intramedullary hardware implantation for bone-anchored limbs due to failed socket use were enrolled in this longitudinal observational cohort study (7 F/7 M, Age: 50.2±12.0 years). The modified Oswestry Disability Index (mODI) (self-reported questionnaire) and whole-body motion capture during overground walking were collected before (with socket prosthesis) and 12-months following bone-anchored limb implantation. Lumbopelvic total range of motion (ROM) and continuous relative phase (CRP) segment angles were calculated during 10 bilateral gait cycles. mODI, total ROM, CRP and CRP variabilities were compared between time points. RESULTS: mODI scores were significantly reduced 12-months after intramedullary hardware implantation for the bone-anchored limb (P = 0.013). Sagittal plane trunk and pelvis total ROM during gait were reduced after implantation (P = 0.001 and P < 0.001, respectively). CRP values were increased (more anti-phase) in the sagittal plane during single limb stance and reduced (more in-phase) in the transverse plane during pre-swing of the amputated limb gait cycle (P << 0.001 and P = 0.029, respectively). No differences in CRP values were found in the frontal plane. SIGNIFICANCE: Decreases in mODI scores and lumbopelvic ROM, paired with the changes in lumbopelvic coordination, indicate that bone-anchored limbs may reduce LBP symptoms and reduce compensatory movement patterns for people with unilateral transfemoral amputation.

Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells.

Cas12a is a promising addition to the CRISPR toolbox, offering versatility due to its TTTV-protospacer adjacent motif (PAM) and the fact that it induces double-stranded breaks (DSBs) with single-stranded overhangs. We characterized Cas12a-mediated genome editing in tomato using high-throughput amplicon sequencing on protoplasts. Of the three tested variants, Lachnospiraceae (Lb) Cas12a was the most efficient. Additionally, we developed an easy and effective Golden-Gate-based system for crRNA cloning. We compared LbCas12a to SpCas9 by investigating on-target efficacy and specificity at 35 overlapping target sites and 57 (LbCas12a) or 100 (SpCas9) predicted off-target sites. We found LbCas12a an efficient, robust addition to SpCas9, with similar overall though target-dependent efficiencies. LbCas12a induced more and larger deletions than SpCas9, which can be advantageous for specific genome editing applications. Off-target activity for LbCas12a was found at 10 out of 57 investigated sites. One or two mismatches were present distal from the PAM in all cases. We conclude that Cas12a-mediated genome editing is generally precise as long as such off-target sites can be avoided. In conclusion, we have determined the mutation pattern and efficacy of Cas12a-mediated CRISPR mutagenesis in tomato and developed a cloning system for the routine application of Cas12a for tomato genome editing.

Matrix metalloproteinases in intestinal fibrosis.

Intestinal fibrosis is a common complication in patients with inflammatory bowel disease [IBD], in particular Crohn's disease [CD]. Unfortunately, at present intestinal fibrosis is not yet preventable, and cannot be treated by interventions other than surgical removal. Intestinal fibrosis is characterized by excessive accumulation of extracellular matrix [ECM], which is caused by activated fibroblasts and smooth muscle cells. Accumulation of ECM results from an imbalanced production and degradation of ECM. ECM degradation is mainly performed by matrix metalloproteinases [MMPs], enzymes that are counteracted by tissue inhibitors of MMPs [TIMPs]. In IBD patients, MMP activity [together with other protease activities] is increased. At the same time, CD patients have a generally lower MMP activity compared to ulcerative colitis patients, who usually do not develop intestinal strictures or fibrosis. The exact regulation and role[s] of these MMPs in fibrosis are far from understood. Here, we review the current literature about ECM remodelling by MMPs in intestinal fibrosis and their potential role as biomarkers for disease progression or druggable targets.

Quality improvement of Dutch ICUs from 2009 to 2021: A registry based observational study.

PURPOSE: To investigate the development in quality of ICU care over time using the Dutch National Intensive Care Evaluation (NICE) registry. MATERIALS AND METHODS: We included data from all ICU admissions in the Netherlands from those ICUs that submitted complete data between 2009 and 2021 to the NICE registry. We determined median and interquartile range for eight quality indicators. To evaluate changes over time on the indicators, we performed multilevel regression analyses, once without and once with the COVID-19 years 2020 and 2021 included. Additionally we explored between-ICU heterogeneity by calculating intraclass correlation coefficients (ICC). RESULTS: 705,822 ICU admissions from 55 (65%) ICUs were included in the analyses. ICU length of stay (LOS), duration of mechanical ventilation (MV), readmissions, in-hospital mortality, hypoglycemia, and pressure ulcers decreased significantly between 2009 and 2019 (OR <1). After including the COVID-19 pandemic years, the significant change in MV duration, ICU LOS, and pressure ulcers disappeared. We found an ICC ≤0.07 on the quality indicators for all years, except for pressure ulcers with an ICC of 0.27 for 2009 to 2021. CONCLUSIONS: Quality of Dutch ICU care based on seven indicators significantly improved from 2009 to 2019 and between-ICU heterogeneity is medium to small, except for pressure ulcers. The COVID-19 pandemic disturbed the trend in quality improvement, but unaltered the between-ICU heterogeneity.

Prognostic Value of Histopathological Thrombus Age in Large Vessel Occlusion-Related Stroke.

INTRODUCTION: Acute mechanical thrombectomy (MT) is the preferred treatment for large vessel occlusion-related stroke. Histopathological research on the obtained occlusive embolic thrombus may provide information regarding the aetiology and pathology of the lesion to predict prognosis and propose possible future acute ischaemic stroke therapy. METHODS: A total of 75 consecutive patients who presented to the Amphia Hospital with acute large vessel occlusion-related stroke and underwent MT were included in the study. The obtained thrombus materials were subjected to standard histopathological examination. Based on histological criteria, they were considered fresh (<1 day old) or old (>1 day old). Patients were followed for 2 years for documentation of all-cause mortality. RESULTS: Thrombi were classified as fresh in 40 patients (53%) and as older in 35 patients (47%). Univariate Cox regression analysis showed that thrombus age, National Institutes of Health Stroke Scale at hospital admission, and patient age were associated with long-term mortality (p < 0.1). Multivariable Cox hazards and Kaplan-Meier analysis demonstrated that after extensive adjustment for clinical and procedural variables, thrombus age persisted in being independently associated with higher long-term mortality (hazard ratio: 3.34; p = 0.038, log-rank p = 0.013). CONCLUSION: In this study, older thromboemboli are responsible for almost half of acute large ischaemic strokes. Moreover, the presence of an old thrombus is an independent predictor of mortality in acute large vessel occlusion-related stroke. More research is warranted regarding future therapies based on thrombus composition.

Pulmonary Surfactant: A Mighty Thin Film.

Pulmonary surfactant is a critical component of lung function in healthy individuals. It functions in part by lowering surface tension in the alveoli, thereby allowing for breathing with minimal effort. The prevailing thinking is that low surface tension is attained by a compression-driven squeeze-out of unsaturated phospholipids during exhalation, forming a film enriched in saturated phospholipids that achieves surface tensions close to zero. A thorough review of past and recent literature suggests that the compression-driven squeeze-out mechanism may be erroneous. Here, we posit that a surfactant film enriched in saturated lipids is formed shortly after birth by an adsorption-driven sorting process and that its composition does not change during normal breathing. We provide biophysical evidence for the rapid formation of an enriched film at high surfactant concentrations, facilitated by adsorption structures containing hydrophobic surfactant proteins. We examine biophysical evidence for and against the compression-driven squeeze-out mechanism and propose a new model for surfactant function. The proposed model is tested against existing physiological and pathophysiological evidence in neonatal and adult lungs, leading to ideas for biophysical research, that should be addressed to establish the physiological relevance of this new perspective on the function of the mighty thin film that surfactant provides.

Dietary supplementation of zinc oxide modulates intestinal functionality during the post-weaning period in clinically healthy piglets.

BACKGROUND: To improve our understanding of host and intestinal microbiome interaction, this research investigated the effects of a high-level zinc oxide in the diet as model intervention on the intestinal microbiome and small intestinal functionality in clinically healthy post-weaning piglets. In study 1, piglets received either a high concentration of zinc (Zn) as zinc oxide (ZnO, Zn, 2,690 mg/kg) or a low Zn concentration (100 mg/kg) in the diet during the post weaning period (d 14-23). The effects on the piglet's small intestinal microbiome and functionality of intestinal tissue were investigated. In study 2, the impact of timing of the dietary zinc intervention was investigated, i.e., between d 0-14 and/or d 14-23 post weaning, and the consecutive effects on the piglet's intestinal functionality, here referring to microbiota composition and diversity and gene expression profiles. RESULTS: Differences in the small intestinal functionality were observed during the post weaning period between piglets receiving a diet with a low or high concentration ZnO content. A shift in the microbiota composition in the small intestine was observed that could be characterized as a non-pathological change, where mainly the commensals inter-changed. In the immediate post weaning period, i.e., d 0-14, the highest number of differentially expressed genes (DEGs) in intestinal tissue were observed between animals receiving a diet with a low or high concentration ZnO content, i.e., 23 DEGs in jejunal tissue and 11 DEGs in ileal tissue. These genes are involved in biological processes related to immunity and inflammatory responses. For example, genes CD59 and REG3G were downregulated in the animals receiving a diet with a high concentration ZnO content compared to low ZnO content in both jejunum and ileum tissue. In the second study, a similar result was obtained regarding the expression of genes in intestinal tissue related to immune pathways when comparing piglets receiving a diet with a high concentration ZnO content compared to low ZnO content. CONCLUSIONS: Supplementing a diet with a pharmaceutical level of Zn as ZnO for clinically healthy post weaning piglets influences various aspects intestinal functionality, in particular in the first two weeks post-weaning. The model intervention increased both the alpha diversity of the intestinal microbiome and the expression of a limited number of genes linked to the local immune system in intestinal tissue. The effects do not seem related to a direct antimicrobial effect of ZnO.

High-throughput sgRNA testing reveals rules for Cas9 specificity and DNA repair in tomato cells.

CRISPR/Cas9 technology has the potential to significantly enhance plant breeding. To determine the specificity and the mutagenic spectrum of SpCas9 in tomato, we designed 89 g(uide) RNAs targeting genes of the tomato MYB transcription factor family with varying predicted specificities. Plasmids encoding sgRNAs and Cas9 were introduced into tomato protoplasts, and target sites as well as 224 predicted off-target sites were screened for the occurrence of mutations using amplicon sequencing. Algorithms for the prediction of efficacy of the sgRNAs had little predictive power in this system. The analysis of mutations suggested predictable identity of single base insertions. Off-target mutations were found for 13 out of 89 sgRNAs and only occurred at positions with one or two mismatches (at 14 and 3 sites, respectively). We found that PAM-proximal mismatches do not preclude low frequency off-target mutations. Off-target mutations were not found at all 138 positions that had three or four mismatches. We compared off-target mutation frequencies obtained with plasmid encoding sgRNAs and Cas9 with those induced by ribonucleoprotein (RNP) transfections. The use of RNPs led to a significant decrease in relative off-target frequencies at 6 out of 17, no significant difference at 9, and an increase at 2 sites. Additionally, we show that off-target sequences with insertions or deletions relative to the sgRNA may be mutated, and should be considered during sgRNA design. Altogether, our data help sgRNA design by providing insight into the Cas9-induced double-strand break repair outcomes and the occurrence of off-target mutations.

Crossing bacterial boundaries: The carbon catabolite repression system Crc-Hfq of Pseudomonas putida KT2440 as a tool to control translation in E. coli.

As a global regulatory mechanism, carbon catabolite repression allows bacteria and eukaryal microbes to preferentially utilize certain substrates from a mixture of carbon sources. The mechanism varies among different species. In Pseudomonas spp., it is mainly mediated by the Crc-Hfq complex which binds to the 5' region of the target mRNAs, thereby inhibiting their translation. This molecular mechanism enables P. putida to rapidly adjust and fine-tune gene expression in changing environments. Hfq is an RNA-binding protein that is ubiquitous and highly conserved in bacterial species. Considering the characteristics of Hfq, and the widespread use and rapid response of Crc-Hfq in P. putida, this complex has the potential to become a general toolbox for post-transcriptional multiplex regulation. In this study, we demonstrate for the first time that transplanting the pseudomonal catabolite repression protein, Crc, into E. coli causes multiplex gene repression. Under the control of Crc, the production of a diester and its precursors was significantly reduced. The effects of Crc introduction on cell growth in both minimal and rich media were evaluated. Two potential factors - off-target effects and Hfq-sequestration - could explain negative effects on cell growth. Simultaneous reduction of off-targeting and increased sequestration of Hfq by the introduction of the small RNA CrcZ, indicated that Hfq sequestration plays a more prominent role in the negative side-effects. This suggests that the negative growth effect can be mitigated by well-controlled expression of Hfq. This study reveals the feasibility of controlling gene expression using heterologous regulation systems.

Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a.

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25-100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.

Adaptor protein complex interaction map in Arabidopsis identifies P34 as a common stability regulator.

Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargoes and coated vesicle accessory proteins. As in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargoes of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.

Testing and evaluation of lower limb prosthesis prototypes in people with a transfemoral amputation: a scoping review on research protocols.

BACKGROUND: When developing new lower limb prostheses, prototypes are tested to obtain insights into the performance. However, large variations between research protocols may complicate establishing the potential added value of newly developed prototypes over other prostheses. OBJECTIVE: This review aims at identifying participant characteristics, research protocols, reference values, aims, and corresponding outcome measures used during prosthesis prototype testing on people with a transfemoral amputation. METHODS: A systematic search was done on PubMed and Scopus from 2000 to December 2020. Articles were included if testing was done on adults with transfemoral or knee disarticulation amputation; testing involved walking with a non-commercially available prototype leg prosthesis consisting of at least a knee component; and included evaluations of the participants' functioning with the prosthesis prototype. RESULTS: From the initial search of 2027 articles, 48 articles were included in this review. 20 studies were single-subject studies and 4 studies included a cohort of 10 or more persons with a transfemoral amputation. Only 5 articles reported all the pre-defined participant characteristics that were deemed relevant. The familiarization time with the prosthesis prototype prior to testing ranged from 5 to 10 min to 3 months; in 25% of the articles did not mention the extent of the familiarization period. Mobility was most often mentioned as the development or testing aim. A total of 270 outcome measures were identified, kinetic/kinematic gait parameters were most often reported. The majority of outcome measures corresponded to the mobility aim. For 48% of the stated development aims and 4% of the testing aims, no corresponding outcome measure could be assigned. Results indicated large inconsistencies in research protocols and outcome measures used to validate pre-determined aims. CONCLUSIONS: The large variation in prosthesis prototype testing and reporting calls for the development of a core set of reported participant characteristics, testing protocols, and specific and well-founded outcome measures, tailored to the various aims and development phases. The use of such a core set can give greater insights into progress of developments and determine which developments have additional benefits over the state-of-the-art. This review may contribute as initial input towards the development of such a core set.

Bronchopneumonia with interstitial pneumonia in beef feedlot cattle: Characterization and laboratory investigation.

Bronchopneumonia with interstitial pneumonia (BIP) has been considered a variant of acute interstitial pneumonia (AIP) rather than a distinct disease. This study compared 18 BIP, 24 bronchopneumonia (BP), and 13 AIP cases in feedlot beef cattle. Grossly, BIP cases typically had cranioventral lung lesions of similar morphology and extent as BP cases, but the caudodorsal lung appeared overinflated, bulged on section, and had interlobular edema and emphysema. Gross diagnosis of BIP had 83% sensitivity and 73% specificity relative to histopathology. Histologic lesions of BIP in cranioventral areas were of chronic BP, while caudodorsal lesions included alveolar and bronchiolar damage and inflammation, interstitial hypercellularity, and multifocal hemorrhages. In BIP cases, cranioventral lung lesions were more chronic than caudodorsal lesions. Histologic scores and microbiology data were comparable in cranioventral lung of BIP versus BP cases and caudodorsal lung of BIP versus AIP cases, with differences reflecting a more chronic disease involving less virulent bacteria in BIP versus BP. Mycoplasma bovis infection was similarly frequent among groups, and a viral cause of BIP was not identified. Lesion morphology and similar blood cytokine concentrations among groups argued against sepsis as a cause of lung injury. Surfactant dysfunction was identified in BIP and BP, and was only partially the result of protein exudation. These and other findings establish BIP as a distinct condition in which chronic cranioventral BP precedes acute caudodorsal interstitial lung disease, supporting a role of chronic inflammation in heightened sensitivity to 3-methylindole or another lung toxicant.

Have Surgery and Implant Modifications Been Associated With Reduction in Soft Tissue Complications in Transfemoral Bone-anchored Prostheses?

BACKGROUND: The most frequently occurring adverse events in individuals with a transfemoral amputation treated with a bone-anchored prosthesis are soft tissue infections and stoma-related complications. These soft tissue complications are believed to be influenced by surgical technique and implant design, but little is known about the effect of changes to treatment on these events. QUESTIONS/PURPOSES: (1) What is the result of surgical technique and implant modifications on the incidence of soft tissue infections and stoma-related complications in transfemoral bone-anchored prosthesis users, depending on whether they had a conventional stoma and a cobalt-chrome-molybdenum (CoCrMo) osseointegration implant (treatment period 2009 to 2013) or a shallower stoma and titanium osseointegration implant (2015 to 2018)? (2) What is the incidence of serious complications, such as bone or implant infection, aseptic loosening, intramedullary stem breakage, and periprosthetic fracture? METHODS: Between 2009 and 2013, we performed osseointegration implant surgery using a conventional surgical technique and a CoCrMo implant in 42 individuals who had a lower extremity amputation experiencing socket-related problems that resulted in limited prosthesis use. We considered all individuals treated with two-stage surgery with a standard press-fit transfemoral osseointegration implant as potentially eligible for inclusion. Based on this, 100% (42) were eligible, and 5% (two of 42) were excluded because they did not provide informed consent, leaving 95% (40 of 42) for analysis. Between 2015 and 2018, we treated 79 individuals with similar indications with osseointegration implant surgery, now also treating individuals with dysvascular amputations. We used an adapted surgical technique resulting in a shallower stoma combined with a titanium implant. Using the same eligibility criteria as for the first group, 51% (40 of 79) were eligible; 49% (39 of 79) were excluded because they were treated with transtibial amputation, a patient-specific implant, or single-stage surgery and 1% (one of 79) were lost before the 2-year follow-up interval, leaving 49% (39 of 79) for analysis. The period of 2013 to 2015 was a transitional period and was excluded from analysis in this study to keep groups reasonably comparable and to compare a historical approach with the present approach. Hence, we presented a comparative study of two study groups (defined by surgical technique and implant design) with standardized 2-year follow-up. The risk factors for adverse events were similar between groups, although individuals treated with the shallow stoma surgical technique and titanium implant potentially possessed an increased risk because of the inclusion of individuals with dysvascular amputation and the discontinuation of prolonged postoperative antibiotic prophylaxis. Outcomes studied were soft tissue infections and stoma-related complications (hypergranulation or keloid formation as well as stoma redundant tissue) and bone or implant infection, aseptic loosening, implant stem breakage, periprosthetic fracture, and death. RESULTS: Patients treated with the shallow stoma surgical technique and titanium implant experienced fewer soft tissue infections (13 versus 76 events, absolute risk 0.17 [95% CI 0.09 to 0.30] versus 0.93 [95% CI 0.60 to 1.45]; p < 0.01), which were treated with less invasive measures, and fewer stoma redundant tissue events (0 versus five events, absolute risk 0 versus 0.06 [95% CI 0.03 to 0.14]) than patients treated with the conventional stoma surgical technique and CoCrMo implant. This was contrasted by an increased incidence of surgical site infections occurring between surgical stages 1 and 2, when no stoma was yet created, after the implementation of treatment changes (conventional surgery and CoCrMo implant versus shallow stoma surgery and titanium implant: one versus 11 events, absolute risk 0.01 [95% CI 0.00 to 0.08] versus 0.14 [95% CI 0.08 to 0.25]; p = 0.02). Patients treated with the shallow stoma surgical technique and titanium implant did not experience serious complications, although bone infections occurred (six events in 8% [three of 40] of patients) in the conventional surgery and CoCrMo implant group, all of which were successfully treated with implant retention. CONCLUSION: Adaptations to surgical technique and newer implant designs, as well as learning curve and experience, have resulted in a reduced incidence and severity of soft tissue infections and stoma redundant tissue, contrasted by an increase in surgical site infections before stoma creation. Serious complications such as deep implant infection were infrequent in this 2-year follow-up period. We believe the benefits of these treatment modifications outweigh the disadvantages and currently advise surgeons to create a shallower stoma with a stable soft tissue envelope, combined with a titanium implant. LEVEL OF EVIDENCE: Level III, therapeutic study.

Growth-coupled enzyme engineering through manipulation of redox cofactor regeneration.

Enzymes need to be efficient, robust, and highly specific for their effective use in commercial bioproduction. These properties can be introduced using various enzyme engineering techniques, with random mutagenesis and directed evolution (DE) often being chosen when there is a lack of structural information -or mechanistic understanding- of the enzyme. The screening or selection step of DE is the limiting part of this process, since it must ideally be (ultra)-high throughput, specifically target the catalytic activity of the enzyme and have an accurately quantifiable metric for said activity. Growth-coupling selection strategies involve coupling a desired enzyme activity to cellular metabolism and therefore growth, where growth (rate) becomes the output metric. Redox cofactors (NAD+/NADH and NADP+/NADPH) have recently been identified as promising target molecules for growth coupling, owing to their essentiality for cellular metabolism and ubiquitous nature. Redox cofactor oxidation or reduction can be disrupted through metabolic engineering and the use of specific culturing conditions, rendering the cell inviable unless a 'rescue' reaction complements the imposed metabolic deficiency. Using this principle, enzyme variants displaying improved cofactor oxidation or reduction rates can be selected for through an increased growth rate of the cell. In recent years, several E. coli strains have been developed that are deficient in the oxidation or reduction of NAD+/NADH and NADP+/NADPH pairs, and of non-canonical redox cofactor pairs NMN+/NMNH and NCD+/NCDH, which provides researchers with a versatile toolbox of enzyme engineering platforms. A range of redox cofactor dependent enzymes have since been engineered using a variety of these strains, demonstrating the power of using this growth-coupling technique for enzyme engineering. This review aims to summarize the metabolic engineering involved in creating strains auxotrophic for the reduced or oxidized state of redox cofactors, and the resulting successes in using them for enzyme engineering. Perspectives on the unique features and potential future applications of this technique are also presented.

Metabolic engineering of Pseudomonas putida KT2440 for medium-chain-length fatty alcohol and ester production from fatty acids.

Medium-chain-length fatty alcohols have broad applications in the surfactant, lubricant, and cosmetic industries. Their acetate esters are widely used as flavoring and fragrance substances. Pseudomonas putida KT2440 is a promising chassis for fatty alcohol and ester production at the industrial scale due to its robustness, versatility, and high oxidative capacity. However, P. putida has also numerous native alcohol dehydrogenases, which lead to the degradation of these alcohols and thereby hinder its use as an effective biocatalyst. Therefore, to harness its capacity as a producer, we constructed two engineered strains (WTΔpedFΔadhP, GN346ΔadhP) incapable of growing on mcl-fatty alcohols by deleting either a cytochrome c oxidase PedF and a short-chain alcohol dehydrogenase AdhP in P. putida or AdhP in P. putida GN346. Carboxylic acid reductase, phosphopantetheinyl transferase, and alcohol acetyltransferase were expressed in the engineered P. putida strains to produce hexyl acetate. Overexpression of transporters further increased 1-hexanol and hexyl acetate production. The optimal strain G23E-MPAscTP produced 93.8 mg/L 1-hexanol and 160.5 mg/L hexyl acetate, with a yield of 63.1%. The engineered strain is applicable for C6-C10 fatty alcohols and their acetate ester production. This study lays a foundation for P. putida being used as a microbial cell factory for sustainable synthesis of a broad range of products based on medium-chain-length fatty alcohols.